This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Drosophila central nervous system (CNS) is composed of a complex array of motoneurons, interneurons and glia. Generation of the mature CNS involves a two step process. First, positional cues across the anterior-posterior and dorsoventral (DV) axes act to specify neural stem cells called neuroblasts. Second, neuroblasts undergo stereotypes cell divisions to generate the precise complement of neuronal and glial cells that populate the mature CNS. Our work focuses on how these positional cues specify the formation of neuroblasts across the DV axis, and how these neuroblasts in turn generate neuronal and glial diversity. Specifically we have shown that three signaling pathways, Dorsal, Egfr and Dpp are required to subdivide the neurectoderm in to three columns across the DV axis, these correspond to the domains of expression of three homeodomain proteins. From ventral to dorsal these are Ventral nervous system defective (Vnd), Intermediate neuroblasts defective (Ind) and Muscle segment homeobox (Msh). Each of these proteins is required for proper formation and/or specification of the corresponding neuroblasts from their respective column. The goal of my research is to establish the link between the dorsoventral restricted homeodomain proteins and the development of the complex array of neurons and glia that compose the Drosophila CNS. The mechanism by which these dorsoventral transcription factors induce different types of neurons and glia is currently unknown in any organism. In order to address this question we have chosen to focus on the role of the Ind homeodomain protein. My lab is currently focused on the characterizing the function of the Ind protein in regulating gene expression. To do this we have identified conserved domains within the protein and are characterizing the function of these domains by testing the function modified versions of the protein in vivo and in cell culture. An alignment on Ind protein sequences from three insect species revealed three domains of highly conserved sequence similarity, including the homeodomain. Preliminary data in cell culture and invivo suggest that the two domains outside the homeodomain are critical for the ability of Ind to act as a transcriptional repressor. We are currently developing an in vivo expression assay that will allow us to characterize the function of these domains in the whole organism. In addition, we have data suggesting that Ind can positively regulate its own expression. To this end, we have shown that in a transcript positive ind mutant ind expression is initiated normally but not maintained. Furthermore we have identified a novel ind regulatory element that contains putative Ind binding sites. We have shown that expression of a reporter gene from this enhancer element is dependent on ind activity, in ind mutant embryos we fail to detect expression. We are currently in the process of further characterization of this element and testing the ability of Ind to regulate transcription from it. To do this we are performing site directed mutagenesis of the putative ind binding sites in order to demonstrate that these sites are required for expression from this construct and that ind binding is necessary. Ultimately we intend to use microarrays to identify novel targets of Ind and Vnd in effort to determine how Ind regulates formation of the intermediate column in the CNS. To this end we have built TAPtagged Ind and Vnd expression constructs we are currently in the process of testing these constructs for their ability to duplicate Ind and Vnd gain of function phenotypes respectively. Preliminary experiments suggest that tagged Ind constructs do function in a manner similar to untagged full length Ind in their ability to repress the known Ind target gene Msh. f the previous projects we are moving forward on identification of Vnd targets with development of new TAPtagged Vnd transgenes. We are currently in the process of characterizing these transgenic fly lines now. We are also attempting to obtain a loss of function phenotype for the CG10479 gene.